Inositolphosphoryl ceramide (IPC) synthase genes from fungi

ABSTRACT

The invention provides isolated nucleic acid compounds encoding IPC synthase or subunit thereof from fungal cells. Also provided are vectors and transformed heterologous host cells for expressing IPC synthase and a method for identifying compounds that inhibit a fungal IPC synthase.

This application claims benefit to U.S. Provisional application Nos.60/043,591, filed Apr. 15, 1997, 60/044,095, filed Apr. 22, 1997,60/046,348, filed May 13, 1997, 60/053,320, filed Jul. 21, 1997,60/062,971, filed Oct. 17, 1997, 60/076,436, filed Mar. 2, 1998.

BACKGROUND OF THE INVENTION

This invention relates to recombinant DNA technology. In particular theinvention pertains to the isolation of novel genes and proteins thatencode inositolphosphoryl ceramide synthase (IPC) synthase or subunitthereof from a variety of fungi and the use of said proteins in screensfor inhibitors of IPC Synthase.

The incidence of life-threatening fungal infections is increasing at analarming rate. With the exception of Staphylococci infections, thefungus C. albicans represents the fastest growing area of concern inhospital acquired bloodstream infections. About 90% of nosocomial fungalinfections are caused by species of Candida with the remaining 10% beingattributable to infections by Aspergillus, Cryptococcus, andPneumocystis. While effective antifungal compounds have been developedfor Candida there is growing concern that the rise in the incidence offungal infections may portend greater resistance and virulence in thefuture. Moreover, anti-Candida compounds frequently do not possessclinically significant activity against other fungal species.

Inositolphosphoryl ceramides are sphingolipids found in a number offungi including but not limited to S. cerevisiae, S. pombe, C. albicans,A. fumigatus, A. nidulans and H. capsulatum. A step of sphingolipidbiosynthesis unique to fungi and plants is catalyzed by the enzyme IPCsynthase. The IPC synthase step, covalently links inositol phosphate andceramide, and is essential for viability in S. cerevisiae. Although someelements of sphingolipid biosynthesis in fungi are shared with mammaliansystems, the pathways diverge at the step after formation of ceramide.Thus, the formation of inositolphosphoryl ceramide is unique to fungiand plants, making IPC synthase a good molecular target for antifungalchemotherapy.

While compounds that target IPC synthase bode well for the future ofanti-fungal therapy, presently there are no clinically useful compoundsthat act at this step. Thus, there is a need for new compounds thatinhibit IPC synthase.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to fungal IPC synthase and to screens forinhibitors thereof.

In one embodiment the invention relates to fungal genes that encode IPCsynthase, or subunit thereof.

In another embodiment, the invention relates to fungal IPC synthasegenes identified herein as SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:10, SEQ ID NO:19, and SEQ ID NO:20.

In another embodiment, the invention relates to nucleic acids that areat least 70% homologous, and/or, will hybridize under high stringencyconditions to a sequence identified herein as SEQ ID NO:1, SEQ ID NO:4,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:19, and SEQ ID NO:20.

In another embodiment the present invention pertains to the proteinsproduced by IPC synthase genes.

In yet another embodiment, the invention relates to proteins designatedherein as SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 8, SEQ ID NO 11, and SEQID NO:21.

In still another embodiment the invention relates to the use of purifiedfungal IPC synthase or subunit thereof in high throughput screens forinhibitors of fungal IPC synthase, said IPC synthase being designatedherein as SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ IDNO:14, SEQ ID NO:17, and SEQ ID NO:21.

In another embodiment the invention relates to the use of recombinanthost cells that carry a vector that expresses a fungal IPC synthase inhigh throughput screens for inhibitors of fungal IPC synthase.

In another embodiment the invention relates to the use of the IPCsynthase genes disclosed herein, or fragments thereof, as hybridizationprobes or PCR primers to identify and isolate homologous genes that arerelated in sequence and/or function.

DEFINITIONS

The terms “cleavage” or “restriction” of DNA refers to the catalyticcleavage of the DNA with a restriction enzyme that acts only at certainsequences in the DNA (viz. sequence-specific endonucleases). The variousrestriction enzymes used herein are commercially available and theirreaction conditions, cofactors, and other requirements are used in themanner well known to one of ordinary skill in the art. Appropriatebuffers and substrate amounts for particular restriction enzymes arespecified by the manufacturer or can readily be found in the literature.

The term “fusion protein” denotes a hybrid protein molecule not found innature comprising a translational fusion or enzymatic fusion in whichtwo or more different proteins or fragments thereof are covalentlylinked on a single polypeptide chain.

The term “plasmid” refers to an extrachromosomal genetic element. Thestarting plasmids herein are either commercially available, publiclyavailable on an unrestricted basis, or can be constructed from availableplasmids in accordance with published procedures. In addition,equivalent plasmids to those described are known in the art and will beapparent to the ordinarily skilled artisan.

“Recombinant DNA cloning vector” as used herein refers to anyautonomously replicating agent, including, but not limited to, plasmidsand phages, comprising a DNA molecule to which one or more additionalDNA segments can or have been added.

The term “recombinant DNA expression vector” as used herein refers toany recombinant DNA cloning vector, for example a plasmid or phage, inwhich a promoter and other regulatory elements are present to enabletranscription of the inserted DNA.

The term “vector” as used herein refers to a nucleic acid compound usedfor introducing exogenous DNA into host cells. A vector comprises anucleotide sequence which may encode one or more protein molecules.Plasmids, cosmids, viruses, and bacteriophages, in the natural state orwhich have undergone recombinant engineering, are examples of commonlyused vectors.

The terms “complementary” or “complementarity” as used herein refers tothe capacity of purine and pyrimidine nucleotides to associate throughhydrogen bonding in double stranded nucleic acid molecules. Thefollowing base pairs are complementary: guanine and cytosine; adenineand thymine; and adenine and uracil.

“Isolated nucleic acid compound” refers to any RNA or DNA sequence,however constructed or synthesized, which is locationally distinct fromits natural location.

A “primer” is a nucleic acid fragment which functions as an initiatingsubstrate for enzymatic or synthetic elongation of, for example, anucleic acid molecule.

The term “promoter” refers to a DNA sequence which directs transcriptionof DNA to RNA.

A “probe” as used herein is a nucleic acid compound that hybridizes withanother nucleic acid compound, and is useful for blot hybridizations,for example. A probe is at least 15 base pairs in length, its sequencebeing at least 90% identical with the nucleic acid molecules disclosedherein, or fragments thereof, or the complements thereof. A probe may ormay not be labeled with a detectable moiety. As used herein, a probe isuseful for hybridization analysis to identify sequences homologous tothose disclosed herein.

The term “hybridization” as used herein refers to a process in which asingle-stranded nucleic acid molecule joins with a complementary strandthrough nucleotide base pairing. “Selective hybridization” refers tohybridization under conditions of high stringency. The degree ofhybridization depends upon, for example, the degree of complementarity,the stringency of hybridization, and the length of hybridizing strands.

The term “stringency” refers to hybridization conditions. Highstringency conditions disfavor non-homologous basepairing. Lowstringency conditions have the opposite effect. Stringency may bealtered, for example, by temperature and salt concentration.

“Low stringency” conditions comprise, for example, a temperature ofabout 37° C. or less, a formamide concentration of less than about 50%,and a moderate to low salt (SSC) concentration; or, alternatively, atemperature of about 50° C. or less, and a moderate to high salt (SSPE)concentration.

“High stringency” conditions comprise a temperature of about 42° C. orless, a formamide concentration of less than about 20%, and a low salt(SSC) concentration; or, alternatively, a temperature of about 65° C.,or less, and a low salt (SSPE) concentration.

“SSC” comprises a hybridization and wash solution. 20X SSC contains 3Msodium chloride, 0.3M sodium citrate, pH 7.0.

“SSPE” comprises a hybridization and wash solution. 1X SSPE contains 180mM NaCl, 9mM Na₂HPO_(4,0.9) mM NaH₂PO₄ and 1 mM EDTA, pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

In yeast and other fungi, for example C. albicans, Aspergillus nidulans,Aspergillus fumigatus, Cryptococcus neofomans, C. Krusei, C.parapsilosis, C. tropicalis, and C. glabrata, IPC synthase catalyzes astep in the synthesis of inositolphosphoryl ceramide from ceramide andphosphatidylinositol (G. Becker and R. Lester, Biosynthesis ofphosphoinositol-containing sphingolipid from phosphatidylinositol by amembrane prepartation from Saccharomyces cervisiae. J. Bacteriol. 142,747-754, 1980). Sphingolipids are necessary for growth and viability ofthe yeast S. cerevisiae. Since IPC synthase is unique to fungi andplants it is a good target for antifungal therapy in mammals.

The IPC synthase gene of C. glabrata comprises the DNA sequencedesignated herein as SEQ ID NO 1. The IPC synthase gene of C. Kruseicomprises the DNA sequence designated herein as SEQ ID NO 4. The IPCsynthase gene of C. parapsilosis comprises the DNA sequence designatedherein as SEQ ID NO 7. The IPC synthase gene of C. tropicalis comprisesthe DNA sequence designated herein as SEQ ID NO 10. The IPC synthasegene of A. fumigatus is designated herein as SEQ ID NO:13. The IPCsynthase gene of A. nidulans is designated herein as SEQ ID NO:16. Thereare no intervening sequences in these genes. The IPC synthase gene of C.neoformans is designated herein as SEQ ID NO:19 and the cDNA thereof asSEQ ID NO:20. There is one intervening sequence in SEQ ID NO:19, frombase pair 1888 through base pair 1939. Those skilled in the art willrecognize that owing to the degeneracy of the genetic code (i.e. 64codons which encode 20 amino acids), numerous “silent” substitutions ofnucleotide base pairs could be introduced into these sequences withoutaltering the identity of the encoded amino acid(s) or protein products.All such substitutions are intended to be within the scope of theinvention. The genes disclosed and contemplated herein are useful forexpressing the protein encoded thereby, in vitro or in a recombinanthost cell.

Also contemplated is the use of said genes and fragments thereof asmolecular hybridization probes for the identification and isolation ofhomologous genes.

Also contemplated by the present invention are nucleic acids thathybridize under high stringency conditions to the nucleic acid sequencesdisclosed herein.

Also contemplated are nucleic acids that are at least 70% identical insequence to a nucleic acid sequence disclosed herein.

Gene Isolation Procedures

Those skilled in the art will recogize that the IPC synthase genesdisclosed herein may be obtained by a plurality of applicable geneticand recombinant DNA techniques including, for example, polymerase chainreaction (PCR) amplification, or de novo DNA synthesis. (See e.g., J.Sambrook et al. Molecular Cloning, 2d Ed. Chap. 14 (1989)).

Skilled artisans will recognize that the IPC synthase genes disclosedherein, or fragments thereof, could be isolated by PCR amplification ofgenomic DNA isolated from suitable fungal cells using oligonucleotideprimers targeted to any suitable region of SEQ ID NO:1, SEQ ID NO:4, SEQID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:19. Theskilled artisan understands that the choice of suitable primers mayinvolve routine experimentation to achieve a successful outcome in a PCRamplification and that some trial and error with specific primers may benecessary. Methods for PCR amplification are widely known in the art.See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Inniset al., Academic Press (1990). The PCR amplification reaction comprisesgenomic DNA, suitable enzymes, primers, and buffers, and is convenientlycarried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk,Conn.). A positive result is determined by detecting anappropriately-sized DNA fragment following agarose gel electrophoresis.

Protein Production Methods

One of the embodiments of the present invention relates to the purifiedproteins encoded by the IPC synthase genes disclosed herein, orfunctionally related proteins.

Skilled artisans will recognize that the proteins of the presentinvention can be synthesized by a variety of different methods. Forexample, the amino acid compounds of the invention can be made bychemical methods, well known in the art, including solid phase peptidesynthesis or recombinant methods. Both methods are described in U.S.Pat. No. 4,617,149, which hereby is incorporated by reference.

Solid phase chemical synthetic methods for polypeptides are well knownin the art, and are described in general texts covering the area. See,e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981)Springer-Verlag, New York, 54-92. For example, peptides may besynthesized by solid-phase methodology utilizing an Applied Biosystems430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) andsynthesis cycles supplied by Applied Biosystems. Protected amino acids,such as t-butoxycarbonyl-protected amino acids, and other reagents arecommercially available from many chemical supply houses.

The proteins of the present invention can also be produced byrecombinant DNA methods using a cloned IPC synthase gene describedherein. Recombinant methods are preferred if a high yield is desired.Expression of a cloned IPC synthase gene can be carried out in a varietyof suitable host cells, well known to those skilled in the art. For thispurpose, an IPC synthase gene is introduced into a host cell by anysuitable means, well known to those skilled in the art. Whilechromosomal integration of the cloned IPC synthase gene is within thescope of the present invention, it is preferred that the gene be clonedinto a suitable extra-chromosomally maintained expression vector so thatthe coding region of the IPC synthase gene is operably-linked to aconstitutive or inducible promoter.

The basic steps in the recombinant production of the IPC synthaseprotein are:

a) constructing a natural, synthetic or semi-synthetic DNA encoding IPCsynthase;

b) incorporating said DNA into an expression vector in a manner suitablefor expressing an IPC synthase protein, either alone or as a fusionprotein;

c) transforming an appropriate eucaryotic or prokaryotic host cell withsaid expression vector,

d) culturing said transformed host cell in a manner to express the IPCsynthase protein; and

e) recovering membranes from said host cell and/or purifying the IPCsynthase protein by any suitable means, well known to those skilled inthe art.

Expressing Recombinant IPC synthase in Procaryotic and Eucaryotic HostCells

In general, prokaryotes are used for cloning DNA sequences and forconstructing the vectors of the present invention. Prokaryotes are alsoemployed in the production of the IPC synthase protein. For example, theEscherichia coli K12 strain 294 (ATCC No. 31446) is particularly usefulfor expressing heterologous proteins in a procaryotic host. Otherstrains of E. coli, bacilli such as Bacillus subtilis,enterobacteriaceae such as Salmonella typhimurium or Serratiamarcescans, various Pseudomonas species and other bacteria, such asStreptomyces, may also be employed as host cells in cloning andexpressing the recombinant proteins of this invention.

Promoters that are suitable for driving expression of genes inprokaryotes include b-lactamase [e.g. vector pGX2907, ATCC 39344,contains a replicon and b-lactamase gene], lactose systems [Chang etal., Nature_(London), 275:615 (1978); Goeddel et al., Nature (London),281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promotersystem [vector pATH1 (ATCC 37695) which is designed to facilitateexpression of an open reading frame as a trpE fusion protein under thecontrol of the trp promoter]. Hybrid promoters such as the tac promoter(isolatable from plasmid pDR540, ATCC-37282) are also suitable. Stillother bacterial promoters, whose nucleotide sequences are generallyknown, enable one of skill in the art to ligate such promoter sequencesto DNA encoding the proteins of the instant invention using linkers oradapters to supply any required restriction sites. Promoters for use inbacterial systems also will contain a Shine-Dalgarno sequenceoperably-linked to the DNA encoding the desired polypeptides. Theseexamples are illustrative rather than limiting.

The proteins of this invention may be synthesized either by directexpression or as a fusion protein comprising the protein of interest asa translational fusion with another protein or peptide, which may beremovable by enzymatic or chemical cleavage. It is often observed in theproduction of certain peptides in recombinant systems that expression asa fusion protein prolongs the lifespan, increases the yield of thedesired peptide, or provides a convenient means of purifying theprotein. A variety of peptidases (e.g. enterokinase and thrombin) whichcleave a polypeptide at specific sites or digest the peptides from theamino or carboxy termini (e.g. diaminopeptidase) of the peptide chainare known. Furthermore, particular chemicals (e.g. cyanogen bromide)will cleave a polypeptide chain at specific sites. The skilled artisanwill appreciate the modifications necessary to the amino acid sequence(and synthetic or semi-synthetic coding sequence if recombinant meansare employed) to incorporate site-specific internal cleavage sites. Seee.g., P. Carter, “Site Specific Proteolysis of Fusion Proteins”, Chapter13, in Protein Purification: From Molecular Mechanisms to Large ScaleProcesses, American Chemical Society, Washington, D.C. (1990).

In addition to prokaryotes, mammalian host cells and eucaryoticmicrobes, such as yeast, may also be used to express the proteins ofthis invention. The simple eucaryote Saccharomyces cerevisiae is themost commonly used eucaryotic microorganism, although a number of otheryeasts, such as Kluyveromyces lactis, are also suitable. For expressionin Saccharomyces, the plasmid YRp7 (ATCC-40053), for example, may beused. See, e.g., D. Stinchcomb et al., Nature, 282:39 (1979); J.Kingsman et al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157(1980). Plasmid YRp7 contains the TRP1 gene which provides a selectablemarker for use in a trp1 auxotrophic mutant.

Purification of Recombinantly-Produced IPC synthase

An expression vector carrying a cloned IPC synthase gene or cDNA fromany of the fungi disclosed herein (SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, or SEQ IDNO:20) is transformed or transfected into a suitable host cell usingstandard methods. Cells which contain the vector are propagated underconditions suitable for expression of the IPC synthase protein. If anIPC synthase gene is under the control of an inducible promoter thengrowth conditions would incorporate the appropriate inducer. Therecombinantly-produced IPC synthase protein may be purified fromcellular extracts of transformed cells by any suitable means. In apreferred method for protein purification, an IPC synthase gene used intransforming a host cell is modified at the 5′ end to incorporateseveral histidine residues at the amino terminus of the encoded IPCsynthase protein. This “histidine tag” enables a single-step proteinpurification method referred to as “immobilized metal ion affinitychromatography” (IMAC), essentially as described in U.S. Pat. No.4,569,794, which hereby is incorporated by reference. The IMAC methodenables rapid isolation of substantially pure protein.

IPC synthase activity can be detected in membranes from recombinantcells transformed with the genes disclosed herein, or in the membranesof non-transformed fungal cells that express said genes. Said membranesare a useful source of IPC synthase activity and can be used as areagent in an assay for IPC synthase activity.

Other embodiments of the present invention relate to isolated nucleicacid sequences which encode SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQID NO:11, SEQ ID NO:14, SEQ ID NO:17, or SEQ ID NO:21 or fragmentsthereof. As skilled artisans will recognize, the amino acid compounds ofthe invention can be encoded by a multitude of different nucleic acidsequences due to the degeneracy of the genetic code. Because thesealternative nucleic acid sequences would encode the same amino acidsequences, the present invention further comprises these alternatenucleic acid sequences.

The IPC synthase genes comprising the present invention may be producedusing synthetic methods well known in the art. See, e.g., E. L. Brown,R. Belagaje, M. J. Ryan, and H. G. Khorana, Methods in Enzymology,68:109-151 (1979). The DNA segments corresponding to an IPC synthasegene could be generated using a conventional DNA synthesizing apparatus,such as the Applied Biosystems Model 380A or 380B DNA synthesizers(Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif.94404) which employ phosphoramidite chemistry. Alternatively,phosphotriester chemistry may be employed to synthesize the nucleicacids of this invention. [See, e.g., M. J. Gait, ed., OligonucleotideSynthesis, A Practical Approach, (1984).]

In an alternative method, IPC synthase DNA sequences comprising aportion or all of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20 can begenerated from fungal genomic DNA using suitable oligonucleotide primerscomplementary to these sequences or region therein, utilizing thepolymerase chain reaction as described in U.S. Pat. No. 4,889,818, whichis incorporated herein by reference. Protocols for performing the PCRare disclosed in, PCR Protocols: A Guide to Method and Applications, Ed.Michael A. Innis et al., Academic Press, Inc. (1990), which hereby isincorporated by reference.

The ribonucleic acids of the present invention may be prepared usingpolynucleotide synthetic methods discussed supra, or they may beprepared enzymatically using RNA polymerases to transcribe a DNAtemplate.

The most preferred system for preparing the ribonucleic acids of thepresent invention employs the RNA polymerase from the bacteriophage T7or bacteriophage SP6. These RNA polymerases are highly specific andrequire the insertion of bacteriophage-specific sequences at the 5′ endof the template to be transcribed. See, J. Sambrook, et al., supra, at18.82-18.84.

This invention also provides nucleic acids, RNA or DNA, which arecomplementary to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ IDNO:22.

The present invention also provides probes and primers useful formolecular biology techniques. A compound that is SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22, or acomplementary sequence thereof, or a fragment thereof, and which is atleast 15 base pairs in length, and which will selectively hybridize toC. glabrata, C. krusei, C. parapsilosis, C. tropicalis or C. neoformansDNA or mRNA encoding IPC synthase, is provided. Preferably, the compoundis DNA.

The probes and primers contemplated herein can be prepared enzymaticallyby well known methods (See e.g. Sambrook et al. supra).

Another aspect of the present invention relates to recombinant DNAcloning vectors and expression vectors comprising the nucleic acidsdescribed and contemplated herein. Many of the vectors encompassedwithin this invention are described above. The preferred nucleic acidvectors are those which comprise DNA. The most preferred recombinant DNAvectors comprise the isolated DNA sequences SEQ ID NO:1, SEQ ID NO:4,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, orSEQ ID NO:20.

The skilled artisan understands that choosing the most appropriatecloning vector or expression vector depends upon a number of factorsincluding the availability of appropriate restriction enzyme sites, thetype of host cell into which the vector is to be transfected ortransformed, the purpose of the transfection or transformation (e.g.,stable transformation as an extrachromosomal element, or integrationinto the host chromosome), the presence or absence of readily assayableor selectable markers (e.g., antibiotic resistance markers, metabolicmarkers, or the like), and the number of copies of the gene to bepresent in the host cell.

Vectors suitable to carry the nucleic acids of the present inventioninclude RNA viruses, DNA viruses, lytic bacteriophages, lysogenicbacteriophages, stable bacteriophages, plasmids, viroids, and the like.The most preferred vectors are plasmids.

When preparing an expression vector the skilled artisan understands thatthere are many variables to be considered, for example, whether to use aconstitutive or inducible promoter. Inducible promoters are preferredbecause they may be the basis for high level and regulatable expressionof an operably-linked gene. The skilled artisan will recognize a numberof inducible promoters and inducers, for example, carbon source, metalions, heat, and others known to the skilled artisan. The practitioneralso understands that the amount of nucleic acid or protein to beproduced dictates, in part, the selection of the expression system. Theaddition of certain nucleotide sequences, such as a sequence encoding asignal peptide preceding the coding sequence, is useful to directlocalization of the resulting polypeptide.

Host cells harboring the nucleic acids disclosed herein are alsoprovided by the present invention. A preferred host is E. coli, intowhich has been transfected or transformed a vector that comprises anucleic acid of the present invention.

The present invention also provides a method for constructing arecombinant host cell capable of expressing SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, or SEQ ID NO:21said method comprising transforming a host cell with a recombinant DNAvector that comprises an isolated DNA sequence that encodes one of thesesequences. Suitable host cells include any strain of E. coli or fungalcell that can accomodate high level expression of a gene(s) introducedby transformation or transfection. Preferred vectors for expression arethose that comprise SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20. Transformedhost cells may be cultured under conditions well known to skilledartisans such that SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:17, or SEQ ID NO:21 is expressed, therebyproducing fungal IPC synthase protein in a recombinant host cell.

For the purpose of identifying or developing antifungal compounds, itwould be desirable to determine those agents which inhibit IPC synthaseactivity. A method for determining whether a substance will inhibit theenzymatic reaction catalyzed by IPC synthase comprises contacting asource of IPC synthase activity (e.g. membrane preparation from a cellthat expresses IPC synthase), or a purified IPC synthase protein, orfragment exhibiting said synthase activity, with a test substance andmonitoring IPC synthase activity by any suitable means.

The instant invention provides such a screening system useful fordiscovering agents which inhibit an IPC synthase, said screening systemcomprising the steps of:

a) preparing a source of IPC synthase, or IPC synthase protein, orsubunit thereof;

b) exposing said IPC synthase or source thereof to a test inhibitor;

c) introducing substrate; and

d) quantifying the loss of activity of said IPC synthase.

Utilization of the screening system described above provides a means todetermine compounds that may interfere with fungal sphingolipidbiosynthesis. This screening system may be adapted to automatedprocedures such as a PANDEX® (Baxter-Dade Diagnostics) system allowingfor efficient high-volume screening of potential therapeutic agents.

In such a screening protocol IPC synthase or subunit thereof is preparedas described herein, preferably using recombinant DNA technology.Alternatively, the reaction can be carried out using membranes fromcells that express IPC synthase, as a source of IPC synthase activity.Preferably, the cells are recombinant cells that incorporate arecombinantly-expressed IPC synthase into the cell membrane. Mostpreferably, the recombinant cells are yeast cells. A sample of a testcompound is then introduced into a reaction vessel containing IPCsynthase activity, followed by the addition of enzyme substrate.Alternatively, substrate may be added simultaneously with the testcompound.

The following examples more fully describe the present invention. Thoseskilled in the art will recognize that the particular reagents,equipment, and procedures described are merely illustrative and are notintended to limit the present invention in any manner.

EXAMPLE 1 Construction of a DNA Vector for Expressing the C. glabrataIPC synthase Gene in Homologous or Heterologous Host

An expression vector suitable for expressing the IPC synthase gene of C.glabrata (SEQ ID NO:1) in E. coli contains an origin of replication(Ori), an ampicillin resistance gene (Amp) useful for selecting cellsthat have incorporated the vector following tranformation. The vectoralso includes the T7 promoter and T7 terminator sequences in operablelinkage to the coding region of the IPC synthase gene. Parent plasmidpET11A (obtained from Novogen, Madison, Wisc.) is linearized bydigestion with appropriate endonucleases and ligated to a DNA fragmentcomprising the coding region of the C. glabrata IPC synthase gene.

The IPC synthase gene ligated into the expression vector is modified atthe 5′ end (amino terminus of encoded protein) in order to simplifypurification of the encoded IPC synthase protein product. For thispurpose, an oligonucleotide encoding 8 histidine residues and a factorXa cleavage site is inserted after the ATG start codon at nucleotidepositions 1 to 3 of SEQ ID NO:1. Placement of the histidine residues atthe amino terminus of the encoded protein enables the IMAC one-stepprotein purification procedure (See below).

EXAMPLE 2 Expression of C. glabrata IPC synthase Gene in Echerichia coliand Purification of IPC synthase Enzyme

A plasmid from Example 1 is transformed into E. coli BL21 (DE3)(hsdS gallcIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (See e.g.Sambrook et al. Supra). Transformants, selected for resistance toampicillin, are chosen at random and tested for the presence of thevector by agarose gel electrophoresis using quick plasmid preparations.Colonies that contain the vector are grown, processed, and the proteinencoded by the IPC synthase gene is purified by immobilized metal ionaffinity chromatography (IMAC), essentially as described in U.S. Pat.No. 4,569,794, the entire contents of which is hereby incorporated byreference.

Briefly, the IMAC column is prepared as follows. A metal-free chelatingresin (e.g. SEPHAROSE 6B IDA, Pharmacia) is washed in distilled water toremove preservative substances and infused with a suitable metal ion[e.g. Ni(II), Co(II), or Cu(II)] by adding a 50 mM metal chloride ormetal sulfate aqueous solution until about 75% of the interstitialspaces of the resin is saturated with colored metal ion. The column isthen ready to receive a crude cellular extract containing the IPCsynthase protein product encoded by the vector.

After removing unbound proteins and other materials by washing thecolumn with suitable buffer, pH 7.5, the bound protein is eluted inbuffer at pH 4.3 essentially as described in U.S. Pat. No. 4,569,794.

EXAMPLE 3 Biochemical Assay for Inhibitors of IPC synthase Using FungalMembrane Preparations

The activity of the IPC synthase enzyme is assayed by preparingmembranes from C. glabrata, for example, and using said membranes as asource of IPC synthase activity.

A suitable, rich medium, for example YEPD, is innoculated with a cultureand allowed to grow overnight at room temperature with vigorous shaking.About 250 ml of fresh medium containing 20 μg/ml myo-inositol isinnoculated with the overnight culture, and grown overnight at 30° C.Cells are harvested by centrifugation and resuspended in ice cold 50 mMpotassium phosphate buffer, pH 7. Cells are washed twice in the samebuffer and then resuspended in the same buffer containing 5 mMdithiothreitol (DTT), 1 μg/ml aprotinin, 0.6 μM leupeptin, 1 mM PMSF,and 1 μg/ml pepstatin A. Cells are ruptured using glass beads in aprocedure that involves 5 successive vortexings each for 30 secondsfollowed by 2 to 5 minute intervals of rest on ice. Membranes arepelleted by centrifugation at 100,000 ×g for 1 hour at 4° C. The pelletis resuspended in cold buffer containing DTT and protease inhibitors anddisrupted further by Dounce homogenization with 5 to 6 strokes on ice.Membranes are mixed with glycerol to a final concentration of 33% andstored frozen at −80° C. For use as “protein stock,” thaw and dilute to4.8 mg/ml in 0.05 M potassium phosphate(Kpi) buffer, pH 7.0 on ice.

IPC synthase is assayed by any suitable method. For example:

Final Assay Conditions:

5-50 μM NBD-C6-ceramide (from Molecular Probes)

1 mM phosphatidylinositol (Sigma-soybean)

2 mM CHAPS

50 mM potassium phosphate buffer, pH 7.0

Up to 2.5% organic co-solvent

Protein to 1.2 mg/ml

Final Vol. 100 μl

2X Master Mix (for 15 samples)

46 μl of 1 mg/ml NBD-C6-ceramide in MeOH

142 μl of 10 mg/ml phosphatidylinositol in CHCl₃

Dry under vacuum Redissolve by adding in order

160 μl 20 mM CHAPS

560 μl deionized water

80 μl 0.5 M Kpi buffer, pH 7.0

Sonicate 5 minutes.

Aliquot 50 μl of 2X master mix to screw cap microfuge at roomtemperature. Add 20 μl of 0.05 M Kpi buffer pH 7.0 to each samplefollowed by 5 μl of test compound or extract. The reaction is initiatedby adding 25 μl of protein stock and incubating for 1 hour in a 30° C.water bath. Reactions are terminated by adding 900 μl of cold absolutemethanol. Samples are stored at −20° C. for 1 hour and centrifuged at14000 ×g at 4° C. for 30 minutes.

The reaction is monitored by HPLC on a Beckman Ultrasphere-ODS 5 μm,150×4 mm column. The chromatogram is developed in 87% MeOH/13% 50 mMTEAP, pH 5.8, at a flow rate of 1 ml/min at room temperature through aBeckman Model 157 Fluorescence detector, usingNBD-C₆-phosphatidylcholine as an internal standard.

Inhibition studies are carried out using the same reaction conditionsdescribed in the preceding paragraph. Compounds to be studied forinhibitory activity are added to final concentrations of between 1 pMand 10 mM.

EXAMPLE 4 Expression of C. parapsilosis (SEQ ID NO:7) IPC synthase Genein S. cerevisiae

A yeast/E.coli shuttle vector suitable for expressing the C.parapsilosis IPC synthase gene (SEQ ID NO:7) in S. cerevisiae isconstructed in parent plasmid YEp351 (See J. Hill et.al., Yeast, 2,163-167, 1986), which contains the multiple cloning region of pUC18, theAmp^(R) gene for selection in E.coli, 2μreplicon, and the LEU2 gene forselection in yeast. A fragment containing the C.parapsilosis IPCsynthase gene is prepared by PCR amplification. Suitable primers to the5′ and 3′ ends of the coding region disclosed in SEQ ID NO:7 areconstructed to contain BamH1 cloning sites in addition to IPC synthasecoding information. After PCR amplification of C.parapsilosis genomicDNA, the amplified fragment is purified by any sutiable method, forexample, gel purification of an appropriately sized fragment, followedby treatment with restriction enzyme BamH1, and ligation to BamH1digested YEp351. The recombinant plasmid carrying the IPC synthase geneis transformed into any suitable leu strain of S. cerevisiae andtransformants selected for growth in a medium that lacks added leucine.Membranes from tranformants are prepared, and IPC synthase activityassayed as in Example 3.

We claim:
 1. An isolated inositolphoshoryl ceramide (IPC) synthaseprotein from a fungal cell comprising the amino acid sequence shown inSEQ ID NO:2.
 2. An isolated inositolphosphoryl ceramide (IPC) synthaseprotein from a fungal cell consisting of the amino acid sequence shownin SEQ ID NO:2.